RhoA is a small GTPase in the Ras homology family exhibiting both GDP/GTP binding and GTPase activities. Like other GTPase molecules, RhoA cycles from an active or GTP-bound conformation to an inactive or GDP-bound conformation by hydrolysis of GTP to GDP. The activation of RhoA plays a key role in Ca+2-independent smooth muscle contraction.
Two isoforms of Rho-kinase, known as Rho-kinase α and β or ROCK II and I, respectively, are important downstream targets of activated RhoA. The α and β isoforms have 64% sequence identity overall and kinase domains that are 90% identical. ROCK I is composed of an N-terminal catalytic kinase domain, a coiled-coil domain, and a C-terminal pleckstrin homology (PH) domain. These domains are common to other closely related kinases, including myotonic dystrophy kinase (DMPK), myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), and citron kinase, except that an additional short coiled-coil domain segment N-terminal to the catalytic domain has been identified in ROCK I (amino acid residues 47-78) and in ROCK II (amino acid residues 63-93) (Amano et al., J. Biol. Chem. 274:32418-32424 (1999)). This short coiled-coil segment is absent in DMPK and MRCK.
Rho-kinases regulate muscle myosin light chain (MLC) proteins both by direct phosphorylation (Kureishi et al., J. Biol. Chem. 272:12257-12260 (1997); Amano et al., J. Biol. Chem. 271:20246-20249 (1996)) and indirectly by phosphorylation of the myosin binding subunit of myosin phosphatase. This phosphorylation inhibits the phosphatase activity, leading to increased levels of phosphorylated MLCs, followed by subsequent muscle contraction (Totsukawa et al., J. Cell. Biol. 150:797-806 (2000)). The Rho/ROCK pathway is also involved in nonmuscle myosin regulation and has been implicated in stress fiber and focal adhesion formation (Ishizaki et al., FEBS Lett. 404:118-124 (1997)); Kawano et al., J. Cell Biol. 147:1023-1038 (1999)), neurite retraction (Amano et al., Genes Cells 3:177-188 (1998)); Hirose et al., J. Cell Biol. 141:1625-1636 (1998)), and tumor cell invasion (Yoshioka et al., J. Biol. Chem. 273:5146-5154 (1998)); Sahai and Marshall, Nat. Cell. Biol. 5, 711-719 (2003)) in nonmuscle cells. Overexpression of RhoA has been associated with colon, breast, lung, and testicular germ cell cancers and in head and neck squamous-cell carcinomas (Sahai, et al., Nature Reviews Cancer 2:133-142 (2002)).
Given the importance of ROCK in regulating such key regulatory processes, structural information on the unique features of the active site of ROCK I would facilitate the discovery of drugs for diseases or disorders in which ROCK plays a role.